The search for new compounds frequently involves screening large libraries of compounds to identify a small subset of compounds that have a desired activity or characteristic. The use of combinatorial chemistry and high-throughput screening has greatly increased the speed at which lead compounds can be identified. Recombinant peptide libraries displayed on phage or other viral particles have proven especially useful in such screens (see, e.g., Cwirla, et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); Devlin, et al., Science 249:404-406 (1990), Scott & Smith, Science 249:386-388 (1990); and Ladner, et al., U.S. Pat. No. 5,571,698, each of which is incorporated herein by reference in its entirety).
Phage display methods typically involve the insertion of random oligonucleotides into a phage genome such that they direct a bacterial host to express peptide libraries fused to phage coat proteins (e.g., filamentous phage pIII, pVI or pVIII). Libraries of up to 1010 individual members can be routinely prepared in this way. Incorporation of the fusion proteins into the mature phage coat results in the peptide encoded by the exogenous sequence being displayed on the exterior surface of the phage, while the exogenous sequence encoding the peptide resides within the phage particle.
This establishment of a physical association between the displayed peptide and the genetic material encoding it allows simultaneous mass screening of very large numbers of phage bearing different peptides. Phage displaying peptides having binding specificity for a particular target can be enriched by affinity screening against the target. The identity of such peptides can be determined from the exogenous sequence contained in the phage displaying the peptide. Peptides so identified can subsequently be synthesized in bulk using conventional synthetic chemistry methods. This technology is further empowered by its very high sensitivity. The ability to amplify hits by culturing phage particles selected in a screen allows a single positive event to be identified.
Phage display also allows screening of peptides in a format in which multiple copies of the same protein are displayed from a single phage. The presence of multiple peptide on the surface allows detection of peptide/target interactions of low affinity. For example, phage display systems in which the peptide is fused to either pIII or pVIII allow the detection of peptides with dissociation constants as high as 100 μM, provided the target is immobilized in active form at high density to permit multivalent bonding of a phage to target molecules.
The basic phage display technology has been expanded to include peptide libraries that are displayed from replicable genetic packages other than phage, such as eukaryotic viruses and bacteria. The principles and strategy are closely analogous to those employed for phage, namely, that nucleic acids encoding peptides to be displayed are inserted into the genome of the package to create a fusion protein between the peptides to be screened and an endogenous protein that is exposed on the cell or viral surface. Expression of the fusion protein and transport to the cell surface results in display of peptides from the cell or viral surface.
A significant limitation with current phage-display technology, is that it is only applicable to display of peptides. Many of the most effective drugs, however, are small organic molecules. Because of the poor pharmacokinetic properties of peptides, lead candidates need to be transformed into non-peptidic structures to fully realize their pharmaceutical potential. A great deal of effort has been dedicated towards converting peptide structures into peptidomimetics, which retain the activity of the peptide but do not suffer from a short serum half-life and poor bioavailability. Unfortunately, these efforts have been largely unsuccessful.